Determination of bone-sialoprotein in bodily fluids for oncological problems

ABSTRACT

The invention relates to antibodies or a plurality of antibodies against human bone sialoprotein (BSP), characterized in that the antibodies bind to epitopes which are present only in human bone sialoprotein from tumor cells, the post-translational glycosylation of which is modified or incomplete in the region of amino acids of 120 to 135, containing the amino acids TGLAA (SEQ ID NO: 2), in comparison with normal bone sialoprotein from bones. The antibodies are put to use in an immunoassay for the diagnosis and prognosis of tumor diseases, in particular the diagnosis and prognosis of bone metastases in the case of primary breast carcinoma.

The present application is a National Stage entry of InternationalApplication No. PCT/EP2002/06219 filed on Jun. 6, 2002, which claimspriority under 35 U.S.C. §119(a)-(d) to European Patent Application01114388.0 filed Jun. 13, 2001.

The invention relates to the immunological determination of bonesialoprotein (BSP) in body fluids, for the diagnosis and monitoring ofdiseases of the bone metabolism and bone structure and in particular forthe diagnosis, prognosis, prophylaxis and therapy of bone metastases ofprimary carcinomas.

The bone sialoprotein (BSP) is a phosphorylated bone glycoprotein havinga relative mass of ca. 80 kDa in the SDS-PAGE. The cDNA for BSP codesfor a peptide sequence of ca. 33 kDa (Fisher L. W. et al. (1990), J.Biol. Chem. 265, 2347-51; U.S. Pat. No. 5,340,934). BSP is one the fewmatrix proteins the occurrence of which on mineralising tissue such asbones, dentin and calcifying cartilage is restricted. BSP represents ca.10 to 15% if the total non-collagenic proteins in the bone matrix. It isas a rule expressed by cells which take part in the formation of dentin,bones and cartilage, for example osteoblasts, developing osteocytes,hypertrophic chondrocytes, odontoblasts and cementoblasts.

Alongside this, BSP is also formed by trophoblasts in the placenta andsome types of cancer cells, e.g. in the case of lungs, breast, prostate,thyroid and neuroblastoma primary and secondary tumors, in the case ofmultiple myeloma and in bone metastases. The degree of expression of BSPby the tumor closely correlates with the severity of the cancer(Waltregny D. et al., Increased expression of bone sialoprotein in bonemetastases compared with visceral metastases in human breast andprostate cancers, in J. Bone Miner. Res., 2000, 15(5), 834-43;Bellahcéne, A. et al., Bone sialoprotein expression in primary humanbreast cancer is associated with bone metastases development, in J. BoneMiner. Res., 1996, 11, 665-670; Waltregny, D. et al., Prognostic valueof bone sialoprotein expression in clinically localised human prostatecancer, in Journal of the National Cancer Institute, 1998, 90,1000-1008; Bellahcéne, A. et al., Expression of bone sialoprotein inprimary breast cancer is associated with poor survival, in Int, J.Cancer, 1996, 69, 350-353).

For dentin, bones and cartilage, two functions are attributed to BSP. Asan adhesion molecule, it is supposed to bring about attachment anddissemination of cells on the tissue matrix. Since in vitro it formscrystallisation nuclei for biological apatite it is suspected that invivo it takes part in mineralisation. The switching off of the BSP genein knock-out mice leads to no recognisable disruption of the buildingand functioning of the skeleton.

In tumors BSP is attributed with participation in microcalcification(Castronovo, V. et al., Evidence that breast cancer associatedmicrocalcifications are mineralized malignant cells, in Int. J. Oncol.,1998, 12, 305-308) and the colonisation of bones by metastasising tumorcells (Bellahcéne, A. et al., Expression of bone sioloprotein in primarybreast caner is associated with poor survival, in Int. J. Cancer, 1996,69, 350-353). The level of concentration of BSP in the serum of patientswith primary carcinomas serves for diagnosis of whether these patientshave bone metastases or such are likely to arise from the primary tumor(Diploma Thesis of Ms. Ina-Alexandra Meier, Development of aradioimmunoassay for the determination of bone sialoprotein (BSP)[“Entwicklung eimes Radioimmunoassays zur Bestimmung vonBonesialoprotein (BSP)]”, 1996, Darmstadt, Technical University[Fachhochschule], Specialist Field Chemical Technology [FB ChemischeTechnologie]; Dissertation of Mr. Markus Karmatschek, Isolation of bonesialoprotein from human bones, Structure of a radioimmunoassay for themeasurement thereof in serum [“Isolierung von Bonesialoprotein aushumanem Kjiochen, Aufbau eines Radioimmunoassays zur dessen Messung imSerum”], 1996; Specialist Field of Biotechnology at the TechnicalUniversity of Darmstadt [F B Biologie der Technischen HochschuleDarmstadt]; Diel I. J. et al., Elevated bone sialoprotein in primarybreast cancer patients is a potent marker for bone metastases; inProceedings of ASCO, 1998, 17, Abstract 461; Diel I. J. et al, Serumbone sialoprotein in patients with primary breast cancer is a prognosticmarker for subsequent bone metastasis, in Clin. Cancer Res., 1999, 5,3914-19; DE 198 13 633; DE 198 21 533; WO 99/50666).

According to recent hypotheses, BSP is supposed to protect thetrophoblasts and BSP-producing tumors from attack by the immune system.Namely, BSP binds with high affinity the factor H of the complementsystem, which is known to restrict the alternative path of thecomplementlysis. Also, BSP can specifically bind to the integrinreceptors on the cell surface through its own recognition sequence(arginine-glycine-aspartate, RGD). In the case of expression of BSP thetumor cells are then supposed to bind the factor H in the blood and inthe tissue fluids to their cell surfaces, or concentrate it around them.Such a protection of BSP from the complement system of the blood of themother is suspected also for the trophoblasts in the placenta (FedarkoN. S. et al. Factor H binding of bone sialoprotein and osteopontinenables tumor cells evasion of complement-mediated attack, in J. Biol.Chem., 200, 275, 16666-16672; WO 00/062065).

Further there is also suspected a function of BSP in angiogenesis. Alongwith the adhesion of osteoclasts and osteoblasts to the bonematrix—through the binding of the RGD recognition sequence in the matrixto the alpha(v)beta(3) integrin receptors on the cell wall—it is alsoobserved that the adhesion, dissemination and orientation of theendothelial cells is probably mediated by BSP. Namely, blood vesselformation around a tumor occurs in parallel with the BSP expression inthe tumor cells (Bellahcéne A et al., Bone sialoprotein mediates humanendothelial cell attachment and migration and promotes angiogenesis, inCirc. Res. 2000, 86(8), 885-91).

BSP thus stands at the center of events in the formation of tumors andmetastases. Thus, the binding of BSP via the RGD sequence to vitronectinor integrin receptors of tumor and epithelial cells can be restricted byantagonists (U.S. Pat. Nos. 6,069,158; 6,008,213; 5,849,865; van derPluijm et al., Bone Sialoprotein peptides are potent inhibitors ofbreast cancer cell adhesion to bone in vitro, in Cancer Res., 1996, 56,1948-1955). EP 1 084 719 A1 teaches a pharmaceutical composition havingBSP as active substance for the support of the repair of damaged boneand connective tissue. WO 94/13310 teaches a composition having a BSPbinding protein of staphylococcus aureaus as active ingredient. WO00/36919 discloses regulatory elements for the purposive monitoring andsuppression of the expression of ESP in tumor and connective tissuecells, which promote calcification.

Since in body fluids free BSP is bound by complement factor H with highaffinity and the BSP can bind to various receptors, its determination isproblematic. Thus, there have been produced in rabbits antibodiesagainst various peptide partial structures of BSP (Fisher, L. W. et al.,Antisera and cDNA probes to human and certain animal model bone matrixnoncollagenous proteins. Acta Orthop Scand Suppl., 1995, 266, 61-655),against recombinant BSP (Stubbs J T 3^(rd) et al., Charaterization ofnative and recombinant bone sialoprotein: delineation of themineral-binding and cell adhesion domains and structural analysis of theRGD domain. J. Bone Miner. Res. 1997 12(8), 1210-22), and against BSPisolated from bones, which antibodies failed to recognise any BSP inhuman serum. Only after separation of the serum proteins via SDS-PAGEcan BSP be detected with these on Western Blots. The significantlylarger factor H molecule of 150 kDa probably masks the smaller BSP (ofca. 65 kDa) to such an extent that antibodies cannot bind. Further,factor H is present in excess in the serum (0.5 mg factor H/mL incomparison to BSP with <20 ng/ml Serum in the case of healthy personsand max. 160 ng/ml in the case of tumor patients). It has been claimedthat due to the binding with factor H immunological direct determinationof BSP in body fluids is impossible, without reducing sample preparation(Fedarko N. S. et al., Factor H binding of bone sialoprotein andosteopontin enables tumor cell evasion of complement-mediated attack, inJ. Biol. Chem., 200, 275, 16666-16672; WO 00/062065). Following ourinvestigations, although such a sample preparation or protein splittingallows a quantitative determination of the BSP present in body fluids,the values obtained allow no answers to be given for oncologicalpurposes.

It is the object of the invention to make available a method for theimmunological determination of BSP in body fluids. It is in particularan object to make available a method for the direct determination of BSPin body fluids. It is a further object of the invention to makeavailable antibodies against BSP for purposes in connection with theprognosis and diagnosis of a remote metastasisation of primarycarcinomas in bones, for the diagnosis of bone metastases and formedical applications.

This object is achieved by means of antibodies according to claim 1 andtheir use for determination of BSP in body fluids. The antibodiesagainst human bone sialoprotein (BSP) can in particular bind epitopeswhich are present on human bone sialoprotein from tumor cells, thepost-translational glycosylation of which is modified or incomplete inthe region of the amino acids 120 to 135 (SWISSPROT: SIAL_HUMAN, Acc.No. P21815, without signal sequence), containing the amino acids TGLAA(SEQ ID NO: 2), in comparison with normal bone sialoprotein from bones.They may be produced with a bone sialoprotein as antigen, preferablywith a bone sialoprotein from tumor cells, which is modified in itsglycosylation chemically or naturally. The bone sialoprotein modified inits glycosylation can also be produced by genetic engineering in tumorcells. It can also be produced against a peptidic antigen, including theamino acid TGLAA (SEQ ID NO: 2) or YTGLAA (SEQ ID NO: 3), if appropriatecoupled to a carrier protein. In a further embodiment, the antibodiesare produced by means of a bone sialoprotein, modified in itsglycosylation, from bone material the donor of which was not capable ofnormal glycosylation of bone proteins. Due to their non-involvement incomplement reactions, IgY antibodies of chicken are particularlypreferred, in particular when these antibodies are human or humanised.

The antibodies in accordance with the invention can be put to use in amethod for the determination of bone sialoprotein of tumor cells in bodyfluids, in particular in serum. In this manner a diagnosis and prognosisof bone metastases can be effected. The antibodies may of course also beused for diagnosis and if applicable for the production of a medicament,either as active ingredient—for example for prophylaxis and therapy ofbone metastases or also as targeting means for the production of adiagnostic means or medicament.

A further object of the invention is diagnostic localisation of tumorsand metastases by means of immune scintigraphy through the antibodies inaccordance with the invention. Thereby the anti-BSP antibodies areradioactively marked and injected into the circulation of the patient.They bind specifically to tumors and metastases tissue and theirdistribution in the body can be pictorially represented then, e.g. bymeans of a scintiscanner.

For binding to the complex of factor H and BSP, the antibody mustrecognise epitopes of BSP which are not masked by the binding partner.The production of such antibodies has previously not been possible. Theinvention makes available such antibodies, because the antibodies aredirected against an isoform of the folded bone sialoprotein (BSP) andbind to epitopes which are formed only by a folded bone sialoproteinfrom tumor cells, the glycosylations of which are modified or incompleteor missing in the region of the amino acids 120 to 135, including theamino acid sequence TGLAA (SEQ ID NO: 2) or YTGLAA (SEQ ID NO: 3), incomparison to the normal bone sialoprotein from bones. Normally therecannot be attained specific antibodies against post-translational orcomplex sugar structures on proteins, since such sugar structures areadded in the same manner and form to many different proteins.Correspondingly, antibodies react against certain sugar structures withmany different proteins and are then considered as a rule to benon-specific and of no value. This is different with bone sialoproteinfrom tumor cells. The altered or missing sugar structure brings about adifferent folding of the bone sialoprotein and creates new epitopes inwhich there are involved both amino acids or peptide structure and alsothe many remaining sugar residues. These epitopes are howevercharacteristic for BSP from degenerate tumor cells.

Antibodies against these epitopes can be produced with a BSP, alteredchemically or naturally in its glycosylation, as antigen and ifapplicable through purification or absorption to the isoform of the boneBSP. Preferably the antibodies are produced with the employment of BSPfrom tumor cells as antigen. Since BSP from tumor cells can be isolatedin sufficient quantities only with difficulty, the geneticallyengineered expression of BSP modified in its glycosylation in tumorcells is the method of choice. It has also been found that some patientshave in the bone material BSP modified in its glycosylation. This meansthat these patients, mostly very old and suffering from seriousosteoporosis, produce a BSP which at least in part is not normallyglycosylated. This BSP also is suitable in principle as an antigen forthe obtaining of the antibodies in accordance with the invention. Theisolation of the partially glycosylated isoform, which is comparable tothe tumor isoform of the BSP, can be carried out analogously todescribed procedures (Karmatschek M et al., Improved purification ofhuman bone sialoprotein and development of a homologousradioimmunoassay, in Clin. Chem. 1997, 43(11), 2076-82).

The antibodies can be produced in mice, guinea pigs, rabbits, dogs,goats, pigs, humans, donkeys or horses, but also in all mammals.Particularly preferred is the immunisation of birds, in particularchicken, since here due to the large evolutionary differences,antibodies against the tumor isoform of BSP can be obtained particularlyeasily. Further, the presence of IgY antibodies does not lead to anactivation of the complement system, which could be problematic due tothe possible binding between factor H and BSP. The antibodies inaccordance with the invention recognise the tumor isoform of BSP in thebonding with factor H.

Thus, subjects of the invention are particular antibodies against theisoforms produced by tumors, and their use for antibody therapy or alsofor immune scintigraphy. As side effects, which are brought about byanti-BSP antibodies, there come in question: direct and indirect damageof the bones and dentin through activation of the immune system againstthe bone matrix and bone cells and/or direct destruction, in theemployment of conjugates of the antibody with cell poisons orradioisotopes. Further, an immune scintigraphy is inconceivable withanti-BSP antibodies which bind to the bone matrix. The matrix would beradioactively marked and the localisation of tumors would be impossible.

The antibodies specific for human BSP are suitable for tumor therapy andlocalisation, since they do not bind or bind to only a slight extent tothe bone matrix or to BSP producing cells of the skeleton and thedentin. In a particularly preferred application of the invention thereare put to use for tumor therapy antibodies which are specific for tumorBSP and additionally recognise BSP in the complex with factor H. Afterapplication of such specific antibodies in tumor patients, free tumorBSP and tumor BSP bonded to factor H, in the blood and in tissue fluid,is neutralised and therewith the protection against complementactivation removed, tumor cells specifically marked for destruction bythe immune system (e.g. through classical activation of the complementcascade) and there are avoided side effects such as e.g. throughactivation of the immune system against the bone matrix or the dentin.In a further application of the invention human polyclonal Anti-BSPantibodies are isolated from the egg of transgenic chickens havinghumanised immune system.

Likewise suitable are monoclonal antibodies from the mouse or thechicken, which fulfil the above-described conditions and which can beobtained by means of screening. In a specific application of the patentthere are employed for this purpose the monoclonal cell lines describedby way of example. Further suitable are Fab fragments obtained throughfragments of antibodies, e.g. proteolytically or by genetic engineering.

Particularly suitable are humanised poly- and monoclonal antibodieswhich recognise BSP in the complex with factor H and do not bind to BSPin the bone matrix. With the application of antibodies of the mouse andof the chicken there is however a particular therapeutic effect to beexpected through formation of human anti-mouse antibodies (HAMA) oranti-chicken antibodies (HACA). HAMAs and HACAs can induce andstrengthen an immune response of the organism to the tumor antigen. Inthe determination of tumor markers there arise, however, interferenceswith the HAMAs and HACAs which disrupt in vitro measuring methods. Inthis manner there are produced falsely high measurement values for tumormarker. This appears after immune scintigraphy or immune therapy withappropriate antibodies, so that a correct tumor marker determination invitro can be effected only after absorption of the HAMAs or HACAs.

These effects can be suppressed through the employment of humanisedantibodies. Polyclonal humanised anti-BSP antibodies can for example beobtained by immunisation of transgenic chickens with BSP, for whichchickens in the embryonic stem cells the gene region for thechicken-specific Fc part of the immunoglobulin (IgY) is exchanged for ahuman specific one (U.S. Pat. No. 5,340,740; U.S. Pat. No. 5,656,479).The humanised antibodies are then deposited in eggs of the chickens andcan be isolated from the egg yolk (Mohammed S. M. et al., Deposition ofgenetically engineered human antibodies into the egg yolk of hens.Immunotechnology, 1998, 4:115-125).

For the production of humanised monoclonal antibodies there may beobtained hybridoma cells of the mouse or the chicken with suitableanti-BSP antibodies, in accordance with standard methods, and from thegenetic material contained in these cells humanised antibodies can bedeveloped through recombination (U.S. Pat. No. 5,585,089; U.S. Pat. No.5,565,332; U.S. Pat. No. 5,225,539; U.S. Pat. No. 5,693,761; U.S. Pat.No. 5,585,089; U.S. Pat. No. 5,530,101).

There will now be described further features and advantages of theinvention with reference to the examples and the accompanying drawings.There is shown:

FIG. 1 a Western Blot with tumor and bone specific isoforms of BSP;

FIG. 2 a Western Blot of the cell culture supernatant of non-transfectedEBNA-293 cells (negative control) and transfected EBNA-293 cells havingthe expression constructs GST-EK-BSP and his₆-myc-EK-BSP with theemployment of a monoclonal mouse anti-BSP antibody;

FIG. 3 the amino acid sequence of secreted BSP (SEQ ID NO: 1) accordingto Fisher et al. (1991);

FIG. 4 an example of a computer printout for a measurement calibrationcurve.

EXAMPLE 1 Characterisation of Tumor and Bone Specific BSP Isoforms inWestern Blot

Serum free supernatants of the human osteosarcoma cell lines UMR-108,MHH-ES 1 and of the breast cancer cell line MCF-7 (oestrogen receptorpositive) and also of human BSP (K-BSP) purified from bones wasseparated by means of SDS-PAGE on a 10% gel under reducing anddenaturing conditions and electrophoretically transferred tonitrocellulose. The membrane was incubated with the monoclonal mouseantibody. The detection of BSP was effected via an anti-mouse antibodyof the goat coupled to peroxidase, and chemoluminescence detection on anX-ray film. The result is shown in FIG. 1. Molecular weight and path ofthe markers are indicated on the left side. The single and doublearrowheads show the different behaviour of the bone/osteosarcoma BSP andMCF-7 BSP. The latter contains additionally a high molecular weight band(triple arrow) which is absent in the other tracks. BSP from one tumorcell line thus has a significantly higher molecular weight than BSP frombone and from osteosarcoma cell lines, whereby beyond this a secondisoform with even higher molecular weight can be observed.

EXAMPLE 2 Production of Polyclonal Antibodies by Means of Immunisationof Chickens with Bone BSP and BSP Peptide Part Structures

Chickens and rabbits were immunised with BSP which was isolated frompatients in accordance with the procedure described by Karmatschek etal.

From the egg yolks and the sera, polyclonal immunoglobulins wereisolated and tested for binding against various peptide part structuresof BSP in an ELISA process. Table 1 shows the results of this epitopemapping. Thereby, peptide part structures of the overall 317 amino acidslong peptide sequence of preproBSP (including leader sequences) werechemically synthesised and bound to a microtitration plate and theantibodies incubated on the plate. The test for binding was effectedafter incubation with a conjugate of peroxidase with anti-IgYimmunoglobulins or anti-rabbit-IgG immunoglobulins and subsequent enzymereaction through transformation of a chromogene as substrate.

TABLE 1 Epitope mapping of the obtained anti-BSP IgG and IgY (SEQ IDNOS: 4-10) Position of the Reaction peptide part strength structure inELISA the BSP Amino acid sequence IgY Rabbit IgG 112-123LeuGlyTyrGlyGluAspAlaThrProGlyThrGly − ? 216-227GluThrGlyGlyGlnGlyLysGlyThrSerLysThr − ? 300-311PheLysGlyGlnGlyTyrAspGlyTyrAspGlyGln − ? 130-144IleGlnLeuProLysLysAlaGlyAspIleThrAsnLysAlaThr +/− + 124-138TyrThrGlyLeuAlaAlaIleGlnLeuProLysLysAlaGlyAsp − ++ 137-151GlyAspIleThrAsnLysAlaThrLysGluLysGluLysGlu- − + SerAspGlu 280-317SerGluAsnGlyGluProArgGlyAspAsnTyrArgAlaTyr- ++ +GluAspGluTyrSerTyrPheLysGlyGlnGlyTyrAspGly-TyrAspGlyGlnAsnTyrTyrHisHisGln Human bone BSP +++ +++

The results show that the obtained chicken antibodies preferentiallybind to the C-terminal sequence of BSP, whereas the rabbit antibodiesbind over a greater region.

Further, polyclonal antibodies (A0001) were obtained by means ofimmunisation of rabbits with the peptide structureTyrThrGlyLeuAlaAlaIleGlnLeuProLysLysAlaGlyAsp (SEQ ID NO: 11, position124-138 of BSP) which preferentially react to this peptide partstructure, but also specifically with human bone BSP.

Polyclonal antibodies (AK_tBSP), however, which were obtained throughimmunisation of rabbits with the peptide part structures ThrGlyLeuAlaAla(SEQ ID NO: 2) (position 125-130), for example TyrThrGlyLeuAlaAla (SEQID NO: 3) (position 124-130), that is after coupling to bovinethyroglobulin as carrier, react with the synthetic peptide partstructure, but not with human bone BSP. These antibodies, surprisingly,recognize exclusively BSP from tumor cells.

For the investigations there were further employed the polyclonalantibodies A002 (obtained from L. W. Fisher) and A003 (obtained from Dr.van Ryden). These antibodies were obtained after immunisation with thepeptide part structures

TyrGluSerGluAsnGlyGluProArgGlyAspAsnTyrArgAlaTyrGluAsp (SEQ ID NO: 12)(A002)

Or

LeuLysArgPheProValGlnGlyGly (SEQ ID NO: 13).

The former peptide originated from the C-terminus of the BSP (positions278-295) and contains the RGD (ArgGlyAsp) recognition sequence of theBSP for receptors of the integrin type. The latter peptide orginatedfrom the N-terminus of the BSP primary structure. Also these peptidespreferentially recognised the respective part structures and reactedspecifically with human bone BSP.

EXAMPLE 3 Production of Recombinant BSP from Breast Cancer Cells, forImmunisation

From the plasmid B6-5g (Fisher L. W. et al., Human bone sialoprotein.Deduced protein sequence and chromosomal localisation, in J. Biol.Chem., 1990, 265(4), 2347-51) the complete cDNA for human BSP (withoutsignal peptide) was amplified by means of PCR and cloned in the episomaleucaryotic expression vector pCEP-Pu (Kohfeldt E et al., Properties ofthe extracellular calcium binding module of the proteoglycan testican,in FEBS Lett. 1997, 414(3), 557-61). The primers were as follows:

Nhe I BSP (sense): (SEQ ID NO: 14) 5′GCCCGCTAGCCTTCTCAATGAAAAATTTGCATCG-3′ Not I BSP (antisense): (SEQ ID NO:15) 5′-CAATGACTGCGGCCGCTCACTGGTGGTGGTAGTAATTC-3″

The Nhe I and Not I slicing sites introduced with the primers werenecessary for the cloning in the expression vector PCEP-PU. This vectoris moreover, for facilitating the protein purification, provided at the5′-ends of the multiple cloning sites with various tags (e.g. His, Myc,G8T). These tags can be detached after purification of the protein witha protease (e.g. factor X or enterokinase) That the correct readingframe was kept to was checked by means of sequencing.

The expression constructs were introduced by means of liposome mediatedstable transfection (FUGENE™ transfection reagent of the company Roche)inter alia into the following human cell lines:

-   -   the embryonic kidney cell line EBNA-293    -   the osteosarcoma cell lines SAOS-2 and MG-63    -   the human breast cancer cell line MCF-7.

A recombinant expression was obtained only in MCF-7 and EBNA-293 cells(see FIG. 2). The osteosarcoma cell lines did not express even afterrepeated transfection attempts.

EXAMPLE 4 Analysis of the Glycosylation of Recombinant BSP fromDegenerate Cells and Bone BSP

Transient cells were cultivated, 48 hours after transfection, for twodays in serum-free medium. So that the proteins in the FCS did not makemore difficult the purification of the recombinant BSP, BSP expressingcells were, after attainment of confluence, cultivated under serum freeconditions. Under these conditions only EBNA-293 cells could survivelonger than 2 to 4. The expression of the recombinant BSP was monitoredthrough SDS-PAGE and immunoblots.

The investigation of serum-free cell culture supernatants yielded withall these cell lines a positive signal in the Western Blot, both withreference to BSP and also the presence of the various tags.

2.5 liter serum-free culture supernatant of the transfected MCF-7 cellline was purified via a nickel Sepharose™ column and there was obtainedtherefrom 250 μg homogeneous His-myc-EK-BSP. The so purified expressionproduct was partially glyglosylated, however had no glycosylation atthreonin 125, that is the threonin in the BSP sequence YT¹²⁵LPAA.

For the glycoanalysis the N-glycanes were enzymatically separated fromthe recombinant BSP (rBSP) or the bone BSP with the peptideN-glycosidase F (PNGase F, Roche). The enzyme brought about a cataylyticsplitting of all N-glycane types from the asparagines. For thedigestion, 20 to 200 μg BSP was precipitated with ethanol and theprecipitant pellet incubated in 1% SDS, β-mercaptoethanol, 0.1 M EDTAfor 30 minutes at room temperature with an excess of enzyme. Therefollowed a digestion with N-glycosidase F overnight at 37° C. Forde-salting the N-glycane solution the digestion was given via a 150 mgcarbon column (carbograph SPE, Alltech) and the N-glycanes eluted with25% aCN in 0.05% TFA.

The O-glycanes were sliced from the BSP by means of water-freehydrazinolysis using a kit (Oglycan release kit, Glyco). For thispurpose, approximately 200 μg salt free BSP was lyophilised for 24hours, had 50 μl hydrazine reagent added thereto under argon protectivegas, dissolved and incubated for 5 hours at 60° C. The hydrazine wasdrawn off under vacuum. There followed a Re-N-acetylisation of theN-acetyl groups with acetic acid anhydrid.

The N- and O-glycanes were marked with the fluorescence dye2-aminobenzamide (Fluka) and the 2-AB marked oligosaccharides digestedsequentially with specific terminal glycosidases and analysed by meansof MALDI-TOF mass spectrometry.

Discussion of the Analysis

The amino acid sequence of human BSP contains four potentialN-glycosylation sites at the positions 88 (NTT), 161 (NGT), 166 (NST)und 174 (NGS). For O-glycosylation there is known no comparableconsensus sequence. All identified N-glycane structures could be foundboth on the BSP isolated from bones and on the recombinant EBNA-293 BSP.There were however differences in the percentage proportion of therespective structures in the total N-glycanes. Thus, the main proportionof the BSP N-glycanes in bones was of triantenary structures (58%) andin the EBNA cell line of tetraantenary structures (48%).

For localisation of the O-glycosylation sites of recombinant BSP, theO-glycanes were removed by means of sequential digestion of the proteinwith neuraminidase, β-galactosidase and β-N-actylhexosaminidase, down tothe core-GaINAc. The partially deglycosylated protein was then split bytreatment with trypsin and V8 protease into peptide fragments. By meansof MALDI-TOF mass spectrometry the masses of the peptides weredetermined and a part of the peptides sequenced by means ofPSD-MALDI-TOF mass spectrometry. With this method, eight O-glycosylationsites of the recombinant BSP could be determined, 5 on the peptide227-245 of SEQ ID NO: 1 (TTTSP . . . QVYR) and a maximum of 3 on thepeptide between AS 120 and AS 135 having the sequence TGLAA (SEQ ID NO:2). Of these, in the recombinant BSP, the threonines in the sequenceDATPGTG (amino acids 117-123 of SEQ ID NO: 1) are O-glycosylated. Withbone BSP there was effected a third O-glycosylation. With recombinantBSP no third glycosylation site is present. Probably, this gylcosylationsite lies on the TGLAA (SEQ ID NO: 2)-BSP part structure.

EXAMPLE 5 Production of Anti-BSP IgY from Egg Yolks

For the purification of greater quantities of anti-BSP IgY for therapyand immune scintigraphy there are described various processes. Theprocess of Akita and Nakai (Akita E. M. et al., Comparison of fourpurification methods for the production of immunoglobulins from eggslaid by hens immunised with an enterotoxigenetic E. coli strain, in JImmunol Methods. 1993, 160(2), 207-14) is preferentially used.

For the egg production there is used a highly productive species such as“Lohmann White” or “Lohmann Brown” with a productivity of 4.5 eggs perweek and a production of over 10 mg specific IgY per yolk. Theimmunisation was effected with BSP antigen isolated from human bones, orrecombinant, in Freund's Adjuvant, whereby after a basic immunisationwith ca. 0.1 mg BSP, booster injections where given every Six weeks.Normally ca. 30% of these chickens do not react to the immunisation. Theeggs were externally disinfected with peracetic acid, then broken andyolk separated from egg white. The yolks were then whisked with 5 to 10times volume ice cold distilled water between pH 5 and 5.2 and incubatedat 2 to 5° C. over 2 to 6 hours. Thereby there sediments out the yolkgranulata which are substantially of lipoproteins. The aqueoussupernatant was then filtered clear through filter paper (e.g. WhatmanNo. 1).

From this supernatant, the anti-BSP IgY can be homogenously purifieddirectly or via affinity chromatography. There was chemically covalentlybonded, through a Sepharose 4B column, activated with cyanogen bromide,BSP isolated from human bones or from culture supernatants ofrecombinant human cell lines. For bonding 1 g IgY there is needed 0.5 gimmobilised BSP (covalently bonded to ca. 5 ml Sepharose™).

The bonded IgY is eluted via an acid gradient and thereafter thesolution neutralised. This solution must then be desalted and theantibodies concentrated, which is possible on an large scale in thecrossflow method (e.g. Amicon™, spiral filter SY 100 with a yield of100,000 Dalton).

EXAMPLE 6 Isolation of anti-BSP IgY which is Bonded to the BSP Factor HComplex

The slight reaction of the polyclonal chicken antibody with BSP in thebone matrix can be excluded through selection of those antibodies whichreact with BSP in the complex with factor H. For this purpose there ischemically covalently bonded through cyanogen bromide activatedSepharose 4B either factor H or BSP isolated from bones or geneticallyengineered, and thereafter so much BSP or factor H applied to the columnand bonded that all ligands in the matrix are complexed with thepartner. Filtered yolk extract is then applied to this affinity columnand as in Example 4 there is now obtained that fraction of antibodieswhich specifically bonds to the free epitope in the BSP-factor Hcomplex.

EXAMPLE 7 Production of Human Anti-BSP Antibodies in Transgenic Chickens

Anti-BSP IgY has in human therapy or diagnosis some weaknesses. Someside effects such as foreign protein reactions are to be expected andthe biological half-life amounts in comparison to human antibodies onlyto 12 to 24 hours. IgY does not activate the complement system.

Human antibodies against BSP can be produced in particular transgenicchickens, in which by means of gene targeting the constant region foravian immunoglobulin in the genes responsible for antibody formation hasbeen exchanged by the constant region for human immunoglobulin. Suitablechicken stem cells and vector systems are described in U.S. Pat. Nos.5,340,740, 5,656,479 and 5,464,764. After immunisation with BSP, suchchickens react with the production of human antibodies in the egg.

EXAMPLE 8 Immunoblot Analysis of the Expression of BSP in Human BreastCancer Cell Lines

The tumor cell lines MDA-MB-231 (breast cancer cell line, oestrogenreceptor negative) MCF-7 (breast cancer cell line, oestrogen receptorpositive) and T-47-D (breast cancer cell line, oestrogen receptorpositive) were extracted with immune precipitation buffer and BSPprecipitated with the polyclonal antibody mixture A0001 of rabbitsagainst human BSP. The precipitates were applied, after denaturing, toSDS gels, the electrophoresis was carried out and the proteinstransferred to nitrocellulose membranes. Thereafter there followed animmune colouring with the anti-BSP rabbit antiserum A001 and amonoclonal mouse-anti-BSP antibody (BSP 1.2), whereby there was employedas second antibody peroxydase conjugates of antibodies of the goatagainst rabbit IgG and against mouse IgG. In both blots A and B thebands of the immune precipitated BSP can be clearly recognized at 70000Dalton.

In order to show the presence or absence of BSP on the cell surface oftumor cells, the cell surfaces of the breast cancer cell linesMDA-MB-231 and MCF-7 were biotinylated, extracted with immuneprecipitation buffer and BSP precipitated with the polyclonal antibodymixture A0001 of the rabbit against human BSP. The precipitates were,after denaturing, applied to SDS gels, the electrophoresis carried outand the proteins applied to a nitrocellulose membrane. Biotinylatedproteins on this membrane were then demonstrated with a conjugate ofperoxydase and streptavidine with the ECL system (Amersham).

EXAMPLE 8a Expression of BSP in and on Breast Cancer Cell Lines

Human breast cancer cells of the lines T-47-D and MDA-MB-231 were markedimmunofluorescently, both with and without prior permeablisation, withan anti-pig-BSP antibody from rabbit and an anti-rabbit antibody of thegoat conjugated with fluorescene. Fluorescently marked BSP can berecognised in both cell lines after permeablisation. Only in the T-47-Dcells could BSP be demonstrated by immunofluorescence also withoutpermeabalisation.

EXAMPLE 9 Detection of BSP Expression in Tumor Cells via RT-PCR

From the tumor cell lines MDA-MB-231 (breast cancer cell line, oestrogenreceptor negative), MCF-7 (breast cancer cell line, oestrogen receptorpositive) and T-47-D (breast cancer cell line, oestrogen receptorpositive) and human fibroblasts (HGF) as control cells, there wasisolated mRNA, by reverse transcriptase the complementary cDNA wasproduced, and the BSP-cDNA amplified by means of PCR with BSP specificprimers. The expression of BSP-mRNA was particularly high in the breastcancer cell line MCF-7, slight in the case of the MDA-MB-231 and T-47-Dcells and not detectable in the control cell line.

EXAMPLE 10 Production of Humanised Monoclonal Antibodies

The monoclonal antibody BSP 1.2 can, due to its specific binding totumor BSP, be put to use for the therapy of primary tumors andmetastases. Thereby, the antibody binds on BSP on the cell surface ofcertain tumor cells and stimulates the immune system to destroy thecells, e.g. via the activation of the complement cascade. Similarly,there can be put to use also the polyclonal or monoclonal anti-BSP IgYfor therapy. When this antibody is used, the human immune system reactswith the formation of its own antibodies—human anti-mouse-IgG antibodies(HAMAs) or human anti-chicken-IgY antibodies (HACAs). HAMAs and HACAscan induce or strengthen an immune response of the organism to the tumorantigen. In the determination of tumor markers there arises, however,interferences with the HAMAs and HACAs which disrupt in vitromeasurement methods. In this way there arises falsely high measurementvalues for tumor marker.

Thus, humanised monoclonal antibodies are particularly suitable for thetherapy and immune scintigraphy. A plurality of methods have beendescribed how one derives appropriately humanised antibodies from thehybridoma cell lines, which produce monoclonal anti-BSP antibodies.

EXAMPLE 11 Conjugates of Anti-BSP Antibodies with Cell Poisons andRadioisotopes

In a further application of the invention there may be chemicallycovalently bonded with the anti-BSP antibodies or their Fab fragmentscell poisons and radioisotopes. Antibodies marked with radioisotopessuch as iodine 125 or iodine 131 are suitable with the application ofsmaller quantities for tumor localisation via immune scintigraphy andwith the application of greater quantities for the direct destruction ofthe tumors. Such chemical conjugates can be produced for example byiodisation of the antibody with iodine 125 or 131 (Garvey, J. S et al.,Methods in Immunology. 3^(rd) ed., W. A. Benjamin Publ., 177, 171-182).An overview of suitable methods for radio immune therapy and immunescintigraphy is found in Vuillez, Radioimmunotargeting: diagnosis andtherapeutic use, in Bull Cancer. 2000, 87(11), 813-27.

EXAMPLE 12 Therapy of Tumors with Expression of BSP on the Cell Surface

It was first determined from biopsy material whether BSP was expressedon the surface of the tumor cells. Patients for whom BSP can be detectedon the surface of the tumor cells can be considered for therapy withanti-BSP antibodies of the chicken, the mouse, the correspondinglyhumanised antibodies and with conjugates of these antibodies with cellpoisons or radioisotopes.

The treatment of tumors with therapeutic antibodies which are directedagainst tumor markers expressed on the cell surface is state of the art.Thus, with the humanised antibody herceptin, against the receptor forthe human epithelial growth factor, breast cancer can be successfullytreated, even in the metastasising form, in ca. 25% of those affected(Hotaling T E et al., The humanized anti-HER2 antibody rhuMAb HER2mediates antibody dependent cell-mediated cytotoxicity via FcgR III[abstract]. Proc Annu Meet Am Assoc Cancer Res 1996; 37:47; Pegram M Det al., Antibody dependent cell-mediated cytotoxicity in breast cancerpatients in Phase III clinical trials of a humanized anti-HER2 antibody[abstract]. Proc Am Assoc Cancer Res 1997; 38:602.

Similarly as with herceptin, the appropriate anti-BSP antibody can beapplied as an infusion, e.g. as a 90 minute infusion in the firstapplication and later as a 30 minute infusion. The frequency of theinfusions and the quantity of the antibodies are determined inaccordance with the half-life of the antibodies in the blood (ca. 6 dayswith a humanised antibody and less than 24 hours with a chickenantibody) and the body weight.

EXAMPLE 13 Therapy of Tumors by Means of Neutralisation of Free BSP, notBonded to Cells, and of the BSP-Factor H Complex

With the methods described above it was determined whether the tumorcells of the patient express BSP which cannot be detected on the cellsurface. In the case of these tumors it can be assumed that the cellsgive out BSP into the blood or the tissue fluid and e.g. through bindingof factor H use this for the inactivation of the alternative path of thecomplement cascade or for migration into bone tissues. A furtherpossible indicator for this tumor type are increased concentrations ofthe BSP in the blood serum (>20 ng/mL serum). In these cases anti-BSPantibodies can be put to use for the neutralisation of the free tumorBSP or the tumor BSP in complexes with factor H. The dose can then beset with regard to the quantity of the BSP present free in the serum andin the tissue fluid. For the therapy, there can be considered anti-BSPantibodies of the chicken, of the mouse and humanised anti-BSPantibodies, which can recognize the free BSP epitope in the complex withfactor H. There can also be considered Fab fragments of theseantibodies, which can be prepared in accordance with a standardprocedure by means of proteolytic digestion (Garvey, J. S et al.,Methods in Immunology. 3^(rd) ed., W. A. Benjamin Publ., 1977, 256-266).Also genetically engineered Fab fragments, derived from the aboveanti-BSP antibodies, come into consideration for such a therapy.

The invention thus makes available antibodies against the human bonesialoprotein (hBSP) which bind specifically only epitopes of hBSP oftumor cells, since tumor hBSP contains no post-translationalO-glycosylation in the region of the amino acids 120 to 135 (SWISSPROT:SIAL_HUMAN, Acc. No. P21815, without signal sequence) containing theamino acids TGLAA (SEQ ID NO: 2). Differently from the normal hBSP frombones. The antibodies can recognize tumorgenic serum hBSP in the complexwith the complement factor H and thus constitute a diagnostic andtherapeutically valuable instrument.

EXAMPLE 14 RIA Measurement Series on Sera of Dialysis and ProstatePatients

The antibodies in accordance with the invention were put to use astrapper antibodies in ELISAs and in comparative measurement series thequantity of BSP in the serum of dialysis patients (“normal” BSP) andprostate patients with individually strongly increased BSP values oftumor origin were investigated.

The serum concentrations of human bone sialoprotein were investigatedwith polyclonal antibodies in accordance with the invention, of chicken,which also bind to epitopes which are present on human bone sialoproteinfrom tumor cells or strongly affected tissues due to the illness, thatis on a human BSP the post-translational glycosylation of which ismodified or incomplete in the region of the amino acids 120 to 135,including the amino acids TGLAA, in comparison with normal bonesialoprotein from healthy bones. As a rule 100 μl of the antibodysolution was mixed with 125I-marked bone sialoprotein. After 24 hourincubation at 4° C., 100 μL solution of a second antibody were added(donkey-anti-chicken-IgG) and after 2 hours incubation at 4° C. thereaction mixture was centrifuged at 2000 G and the supernatant removed.After washing with 250 μL in PBS and a subsequent centrifuging (10 minat 2000G) the supernatant was again removed and the radioactivity of thepellets after addition of a counter fluid determined for 1 minute with agamma-counter.

The necessary calibration curve was produced in the normal way, in thiscase with the aid of a 4-parameter curve algorithm. The standard valuescontained as a rule 0, 1.9, 3.8, 7.5, 15, 30, 60 and 120 μg/L BSP. Theevaluation of the analytical accuracy through the addition ofcalibrators to the patient samples yielded as a mean a recovery in therange of over 99%. The computer printout for an example calibrationcurve is shown in FIG. 4.

The tabular measurement series below show that with the aid of theantibodies in accordance with the invention there can be reliablydetermined tumor and non-modified BSP in the serum of patients,quantitatively, and this in the presence of factor H and independentlyof the specific disease symptoms of the patients. A difficult and errorprone separation of the serum proteins such as factor H from BSP is notnecessary. In particular BSP from tumor cells can be reliablydetermined, which brings with it significant advances in the prognosisand the characterisation of the primary tumors and possible metastases.

TABLE 2 Measurement series of serum of patients with prostate carcinomaSam- ID-No: ple Sample Sialoprotein Box 1-4 Sialo No. Code Diagn volumes(ig/L) 1 1 182 E096 7 81.2 1 2 183 E162 7 479.0 1 3 184 E278 7 28.0 1 4185 E476 7 10.8 1 5 186 E416 7 38.5 1 6 187 E560 7 3.9 1 7 188 H180 730.2 1 8 189 E653 7 344.7 1 9 190 D268 7 112.3 1 10 191 E527 7 17.4 1 11192 E524 7 18.5 1 12 193 E441 7 72.2 1 13 194 H096 7 251.0 1 14 195 E8247 33.4 1 15 196 E971 7 measurement ranges 1 16 197 D411 7 10.9 1 17 198H601 7 41.1 1 18 199 H567 7 130iL measurement ranges 1 19 272 H280 731.5 1 20 273 H316 7 1 21 274 H420 7 238.9 1 22 275 I006 7 17.9 1 23 276I007 7 88.0 1 24 277 I033 7 27.2 1 25 278 I084 7 80.2 1 26 279 I093 729.1 1 27 280 I142 7 447.0 1 28 281 9407(Mü) 7 252.0 1 29 282 9245(Mü) 737.3 1 30 283 8940(Mü) 7 250iL 102.9 1 31 284 8779(Mü) 7 1 32 2858712(Mü) 7 11.9 1 33 286 8689(Mü) 7 7.4 1 34 287 8400(Mü) 7 32.7 1 35288 8388(Mü) 7 30.8 1 36 289 8279(Mü) 7 1 37 290 H125 7 measurementranges 1 38 291 H172 7 9.0 1 39 292 H180 7 — 1 40 293 H487 7 5.9 1 41104 F968 4 3.2 1 42 105 F965 4 76.7 1 43 106 F923 4 8.4 1 44 107 F908 411.1 1 45 108 F821 4 17.7 1 46 109 F836 4 3.5 1 47 110 F342 4 3.0 1 48111 F214 4 8.4 1 49 112 G843 4 6.7 1 50 113 G777 4 10.9 1 51 114 G753 46.8 1 52 115 G381 4 3.1 1 53 116 G984 4 32.1 1 54 117 G891 4 17.0 1 55118 F196 4 3.4 1 56 119 F183 4 6.9 1 57 120 G010 4 20.6 1 58 121 F453 46.0 1 59 122 F640 4 2.6 1 60 123 G462 4 10.7 1 61 124 F423 4 4.7 1 62125 F360 4 3.0 1 63 126 F875 4 7.5 2 64 127 G798 4 8.9 2 65 128 G786 44.9 2 66 129 G384 4 8.2 2 67 130 F529 4 7.1 2 68 131 E405 4 1.3 2 69 132E419 4 2 70 133 E274 4 17.2 2 71 134 E545 4 6.3 2 72 135 E166 4 14.5 273 136 F601 4 8.0 2 74 137 G432 4 5.3 2 75 138 F264 4 4.5 2 76 139 D2974 2 77 140 E355 4 180iL 8.9 2 78 141 E950 4 10.0 2 79 142 E391 4 8.3 280 69 F053 3 11.0 2 81 70 F177 3 6.5 2 82 71 F068 3 8.0 2 83 72 F237 33.6 2 84 73 F255 3 7.5 2 85 74 F378 3 2.1 2 86 75 F471 3 7.5 2 87 76F484 3 6.0 2 88 77 F555 3 12.5 2 89 78 F281 3 7.0 90 79 F287 3 4.0 2 9180 F372 3 3.8 2 92 81 F496 3 2 93 82 F536 3 4.2 2 94 83 F561 3 9.9 2 9584 F573 3 2 96 85 F604 3 6.6 2 97 86 F651 3 5.4 2 98 87 F689 3 1.7 2 9988 F695 3 5.7 2 100 89 F728 3 8.8 2 101 90 F785 3 200iL 3.0 2 102 91F797 3 5.3 2 103 92 F824 3 3.3 2 104 93 F005 3 220iL 2.6 2 105 94 F017 33.0 2 106 95 F866 3 6.3 2 107 96 F881 3 2 108 97 F890 3 6.7 109 98 F9353 5.7 2 110 99 F944 3 6.7 2 111 100 F947 3 8.0 2 112 101 F950 3 2 113102 F983 3 8.9 2 114 103 F986 3 5.7 2 115 1 H128 1 5.2 2 116 2 H073 18.9 2 117 3 H072 1 18.8 2 118 4 H131 1 6.9 2 119 5 H082 1 5.3 2 120 6H052 1 3.2 2 121 7 H057 1 120iL 5.2 3 122 8 H060 1 2.7 3 123 9 H061 12.7 3 124 10 H062 1 2.0 3 125 11 H063 1 3.0 3 126 12 H065 1 4.3 3 127 13H067 1 3.9 3 128 14 H070 1 120iL 4.3 3 129 15 H097 1 3.8 3 130 16 H116 1220iL 11.0 3 131 17 H134 1 3.9 3 132 18 H139 1 3.7 3 133 19 H140 1 12.13 134 20 H143 1 7.2 3 135 21 H163 1 5.1 3 136 22 H051 1 3.1 3 137 23H244 1 6.7 3 138 24 H271 1 2.6 3 139 25 H006 1 4.7 3 140 26 H250 1 3.5 3141 27 H389 1 260.0 3 142 28 H302 1 2.9 3 143 29 H336 1 8.0 3 144 30H004 1 11.8 3 145 31 G619 1 — 146 32 D749 1 8.7 3 147 33 D761 1 6.6 3148 34 1 1 4.2 3 149 35 16 1 8.5 3 150 36 G969 1 27.2 3 151 148 E032 69.8 3 152 149 E938 6 9.4 3 153 150 E674 6 210iL 11.5 3 154 151 E890 610.5 3 155 152 E082 6 27.7 3 156 153 E395 6 210iL 13.9 3 157 154 E668 627.9 3 158 155 E242 6 3 159 156 G320 6 7.6 3 160 157 G248 6 9.9 3 161158 G055 6 14.5 3 162 159 G034 6 5.8 3 163 160 G804 6 7.2 3 164 161 G7206 6.2 3 165 162 G774 6 10.6 166 163 G699 6 2.7 3 167 164 G672 6 9.0 3168 F776 6 15.5 3 169 166 F869 6 4.3 3 170 167 H169 6 8.9 3 171 F199 613.2 3 172 169 F246 6 46.0 3 173 170 F502 6 7.4 3 174 171 E806 6 7.5 3175 172 E929 6 18.2 3 176 173 E494 6 6.7 3 177 174 E758 6 6.8 3 178 175E692 6 15.4 3 179 176 G025 6 19.3 3 180 177 G723 6 16.0 3 181 178 F508 632.7 4 182 179 G227 6 8.1 4 183 180 H261 6 23.8 4 184 181 H314 6 28.0 4185 294 H599 7 — 4 186 295 H601 7 — 4 187 296 H737 7 7.6 4 188 297 H9557 14.6 4 189 298 H980 7 10.0 4 190 299 I010 7 3.7 4 191 300 I282 7 18.04 192 301 9742(Mü) 7 42.0 4 193 302 9773(Mü) 7 22.3 4 194 303 9788(Mü) 715.7

The diagnosis numbers stand for:

1=healthy men

3=BPH

4=PCa pN0 M0

6=PCa pN1 M0

7=PCa pN1 M1 ossae

The pathomorphological classification of the carcinomas was effected inaccordance with Van Nuys. M stands for metastases.

TABLE 3 determination of BSP in serum of dialysis patients SamplePatient BSP Remark: No. Code ng/ml Serum Apparatus sequence L50 G**.E.18.8 10 Serum > AxSym1(alt): B/10 L69 H**.R. 10.1 10 Serum >AxSym1(alt): F/07 L85 W**.D 77.3 10 Serum > HITACHI 917: 5006/1 L131K**.E. 17.1 10 Serum > AxSym1(alt): A/09 L13 B**.M. 36.6 10 Serum >AxSym1(alt): A/04 L14 H**.L. 6.6 10 Serum > AxSym1(alt): C/04 L15 P**.B.8.1 10 Serum > AxSym1(alt): C/02 L16 S**.I. 10.4 10 Serum > AxSym1(alt):E/09 L24 H**.A. 28.1 10 Serum > AxSym1(alt): B/04 L38 B**.M. 3.1 10Serum > AxSym1(alt): A/03 L41 K**.C. 12.5 10 Serum > AxSym1(alt): B/02L50 H**.L. 31.9 10 Serum > AxSym1(alt): C/03 L77 W**.P. 7.8 10 Serum >AxSym1(alt): A/04 L84 D**.K. 46.9 10 Serum > AxSym1(alt): D/08 L88M**.H. 26.5 10 Serum > AxSym2(neu): A/01 L134 S**.M. 4.2 10 Serum >AxSym1(alt): E/04 L73 H**.K. 5.2 10 Serum > AxSym1(alt): D/01 L75 L**.I.5.5 10 Serum > AxSym1(alt): B/05 L78 Z**.U. 15.2 10 Serum > AxSym1(alt):A/01 L79 P**.B. 3.2 10 Serum > AxSym1(alt): B/01 L82 G**.B. 29.7 10Serum > AxSym1(alt): A/02 L85 S**.I. 18.0 10 Serum > AxSym1(alt): E/04L91 K**.H. 12.4 10 Serum > AxSym1(alt): C/07 L93 S**.G. 17.0 10 Serum >AxSym1(alt): A/02 L96 S**.M. 8.9 10 Serum > AxSym1(alt): A/07 L113S**.G. 18.9 10 Serum > AxSym1(alt): A/04 L114 R**.H. 22.7 10 Serum >AxSym1(alt): A/05 L117 R**.A. 16.0 10 Serum > AxSym1(alt): A/09 L53L**.E. 44.4 10 Serum > AxSym1(alt): A/08 L71 M**.M. 11.4 10 Serum >AxSym1(alt): B/04 L94 W**.E. 19.0 10 Serum > AxSym1(alt): B/04 L10S**.L. 12.3 10 Serum > AxSym1(alt): E/03 L12 N**.J. 5.0 10 Serum >AxSym1(alt): D/02 L13 C**.F: 7.0 10 Serum > AxSym1(alt): D/04 L17 L**.I.6.7 10 Serum > AxSym1(alt): A/05 L18 S**.R. 8.7 10 Serum > AxSym1(alt):A/06 L19 L**.E. 11.0 10 Serum > AxSym1(alt): A/09 L24 H**.F. 18.6 10Serum > AxSym1(alt): A/05 L43 H**.L. 17.8 10 Serum > AxSym1(alt): B/06L45 S**.C. 3.1 10 Serum > AxSym1(alt): B/07 L1 S**.W. 21.5 10 Serum >HITACHI 917: 7002/1 L2 S**.M. 14.2 10 Serum > HITACHI 917: 7020/2 L3S**.A. 11.8 10 Serum > AxSym1(alt): A/02 L4 D**.J. 14.4 10 Serum >AxSym1(alt): A/01 L5 G**.H. 9.0 10 Serum > AxSym1(alt): A/02 L6 B**.U.4.7 10 Serum > AxSym1(alt): A/03 L7 S**.L 8.2 10 Serum > AxSym1(alt):A/01 L8 H**.E. 15.3 10 Serum > AxSym1(alt): A/04 L9 N**.H. 7.1 10Serum > AxSym1(alt): E/03

An increased BSP value in the serum (>20-25 μg/L) here indicates that apathological alteration of the bone structure or an increased bonemetabolism is present.

1. Purified antibodies that specifically bind epitopes present in humanbone sialoprotein (BSP) produced in tumor cells and absent in BSPproduced in normal bone cells, wherein the post-translationalglycosylation of BSP produced in tumor cells is modified or incompletein the region of amino acids 120 to 135 of SEQ ID NO: 1 in comparisonwith the post-translational glycosylation of human BSP produced innormal bone cells, and wherein the epitopes comprise the amino acidsequence TGLAA (SEQ ID NO: 2) or YTGLAA (SEQ ID NO: 3).
 2. Antibodiesaccording to claim 1, produced against a peptidic antigen that includesthe amino acid sequence TGLAA (SEQ ID NO: 2) or YTGLAA (SEQ ID NO: 3),said peptidic antigen being optionally coupled to a carrier protein. 3.Antibodies according to claim 1 or 2, which are human or humanized.
 4. Apharmaceutical composition comprising as active ingredient antibodiesaccording to claim 1 or
 2. 5. A method for the determination of bonesialoprotein from tumor cells in body fluids, comprising contacting asample from said body fluid with antibodies according to claim 1 or 2and detecting binding of said antibodies to bone sialoprotein from saidsample.
 6. The method according to claim 5 for the diagnosis andprognosis of bone metastases.